Analysis of covalent flavinylation using thermostable succinate dehydrogenase from Thermus thermophilus and Sulfolobus tokodaii lacking SdhE homologs

Recent studies have indicated that post-translational flavinylation of succinate dehydrogenase subunit A (SdhA) in eukaryotes and bacteria require the chaperone-like proteins Sdh5 and SdhE, respectively. How does covalent flavinylation occur in prokaryotes, which lack SdhE homologs? In this study, I showed that covalent flavinylation in two hyperthermophilic bacteria/archaea lacking SdhE, Thermus thermophilus and Sulfolobus tokodaii, requires heat and dicarboxylic acid. These thermophilic bacteria/archaea inhabit hot environments and are said to be genetically far removed from mesophilic bacteria which possess SdhE. Since mesophilic bacteria have been effective at covalent bonding in temperate environments, they may have caused the evolution of SdhE.     

Evaluation of a novel thermo-alkaline Staphylococcus aureus lipase for application in detergent formulations

An extracellular lipase of a newly isolated S. aureus strain ALA1 (SAL4) was purified from the optimized culture medium. The SAL4 specific activity determined at 60 °C and pH 12 by using olive oil emulsion or TC4, reached 7215 U/mg and 2484 U/mg, respectively. The 38 NH₂-terminal amino acid sequence of the purified enzyme starting with two extra amino acid residues (LK) was similar to known staphylococcal lipase sequences. This novel lipase maintained almost 100% and 75% of its full activity in a pH range of 4.0–12 after a 24 h incubation or after 0.5 h treatment at 70 °C, respectively. Interestingly, SAL4 displayed appreciable stability toward oxidizing agents, anionic and non-ionic surfactants in addition to its compatibility with several commercial detergents. Overall, these interesting characteristics make this new lipase promising for its application in detergent industry.     

Genome sequence of the phage clP1, which infects the beer spoilage bacterium Pediococcus damnosus

Pediococcus damnosus (P. damnosus) bacteriophage (phage) clP1 is a novel virulent phage isolated from a municipal sewage sample collected in Southern Ireland. This phage infects the beer spoilage strain P. damnosus P82 which was isolated from German breweries. Sequencing of the phage has revealed a linear double stranded DNA genome of 38,013 base pairs (bp) with an overall GC content of 47.6%. Fifty seven open reading frames (ORFs) were identified of which 30 showed homology to previously sequenced proteins, and as a consequence 20 of these were assigned predicted functions. The majority of genes displayed homology with genes from the Lactobacillus plantarum phage phiJL-1. All genes were located on the same coding strand and in the same orientation. Morphological characterisation placed phage clP1 as a member of the Siphoviridae family with an isometric head (59 nm diameter) and non-contractile tail (length 175 nm; diameter 10nm. Interestingly, the phage clP1 genome was found to share very limited identity with other phage genome sequences in the database, and was hence considered unique. This was highlighted by the genome organisation which differed slightly to the consensus pattern of genomic organisation usually found in Siphoviridae phages. With the genetic machinery present for a lytic lifecycle and the absence of potential endotoxin factors, this phage may have applications in the biocontrol of beer spoilage bacteria. To our knowledge, this study represents the first reported P. damnosus phage genome sequence.     

sNASP, a histone H1-specific eukaryotic chaperone dimer that facilitates chromatin assembly

NASP has been described as a histone H1 chaperone in mammals. However, the molecular mechanisms involved have not yet been characterized. Here, we show that this protein is not only present in mammals but is widely distributed throughout eukaryotes both in its somatic and testicular forms. The secondary structure of the human somatic version consists mainly of clusters of α-helices and exists as a homodimer in solution. The protein binds nonspecifically to core histone H2A-H2B dimers and H3-H4 tetramers but only forms specific complexes with histone H1. The formation of the NASP-H1 complexes is mediated by the N-and C-terminal domains of histone H1 and does not involve the winged helix domain that is characteristic of linker histones. In vitro chromatin reconstitution experiments show that this protein facilitates the incorporation of linker histones onto nucleosome arrays and hence is a bona fide linker histone chaperone.     

Modulatory efficacy of green tea polyphenols on glycoconjugates and immunological markers in 4-Nitroquinoline 1-oxide-induced oral carcinogenesis-A therapeutic approach

Green tea polyphenols (GTP) has been used as a chemopreventive agent world wide against chemically induced cancer. The present study is aimed to understand the therapeutic action of GTP on glycoconjugates and immunological markers in 4-Nitroquinoline 1-oxide (4-NQO)-induced oral cancer over a period of 30 days at 200mg/kg, p.o., Oral cancer was induced by painting 4-NQO for 8 weeks followed by administration of GTP after 22 weeks, for 30 days. Glycoconjugates such as hexose, hexosamine, sialicacid, fucose and mucoprotein were analysed. Expression of glycoconjugates was examined through histology and SDS-PAGE. Immunological markers such as circulating immune complex and mast cell density were studied. Oral cancer-induced animals showed a significant increase in levels of glycoconjugates and its expression, similar to that observed for immunological markers. Treatment with GTP altered the expression of glycoconjugates as well as immunological markers. The results suggest that GTP modulates both the expression of glycoconjugates and immunological markers resulting in regression of oral cancer.     

Expression and characterization of a recombinant Cry1Ac crystal protein fused with an insect-specific neurotoxin ω-ACTX-Hv1a in Bacillus thuringiensis

In order to assess possible enhancement of biopesticide activity, the fusion gene of crystal protein gene cry1Ac with the insect-specific neurotoxin ω-ACTX-Hv1a gene and egfp was expressed in Bacillus thuringiensis acrystalliferous strain Cry-B under the control of the native gene expression system. The fusion recombinant Cry-B(1Ac-ACTX-EGFP) generally produced two or three small crystal-like inclusion bodies in each cell and the GFP signal could be clearly observed. A 166 kDa full-length fusion protein was identified by immunoblot analysis. Virulence of the fusion inclusions was at least fivefold higher toward larvae of Spodoptera exigua. These results demonstrated that a foreign protein could be expressed and accumulate as parasporal inclusions in B. thuringiensis by C-terminal fusion with the native endotoxin while retaining partial insecticidal activity.     

Solid-state NMR spectroscopy of 18.5 kDa myelin basic protein reconstituted with lipid vesicles: Spectroscopic characterisation and spectral assignments of solvent-exposed protein fragments

Myelin basic protein (MBP, 18.5 kDa isoform) is a peripheral membrane protein that is essential for maintaining the structural integrity of the multilamellar myelin sheath of the central nervous system. Reconstitution of the most abundant 18.5 kDa MBP isoform with lipid vesicles yields an aggregated assembly mimicking the protein's natural environment, but which is not amenable to standard solution NMR spectroscopy. On the other hand, the mobility of MBP in such a system is variable, depends on the local strength of the protein-lipid interaction, and in general is of such a time scale that the dipolar interactions are averaged out. Here, we used a combination of solution and solid-state NMR (ssNMR) approaches: J-coupling-driven polarization transfers were combined with magic angle spinning and high-power decoupling to yield high-resolution spectra of the mobile fragments of 18.5 kDa murine MBP in membrane-associated form. To partially circumvent the problem of short transverse relaxation, we implemented three-dimensional constant-time correlation experiments (NCOCX, NCACX, CONCACX, and CAN(CO)CX) that were able to provide interresidue and intraresidue backbone correlations. These experiments resulted in partial spectral assignments for mobile fragments of the protein. Additional nuclear Overhauser effect spectroscopy (NOESY)-based experiments revealed that the mobile fragments were exposed to solvent and were likely located outside the lipid bilayer, or in its hydrophilic portion. Chemical shift index analysis showed that the fragments were largely disordered under these conditions. These combined approaches are applicable to ssNMR investigations of other peripheral membrane proteins reconstituted with lipids.     

Vortex-Induced Formation of Insulin Amyloid Superstructures Probed by Time-Lapse Atomic Force Microscopy and Circular Dichroism Spectroscopy

Misfolding and aggregation of proteins are multipathway processes that result in polymorphism of amyloid fibrils. While agitation is one of the most common means of inducing structural variants of fibrils (the so-called ‘amyloid strains’), there is as yet no mechanistic explanation for this effect. In this study, time-lapse atomic force microscopy has been employed to probe insulin fibrillation upon intensive agitation. At 60 °C, the initial stages of aggregation in agitated samples are similar to those in quiescent solutions; however, in vortexed samples, an abrupt and highly cooperative collapse of early filaments occurs, yielding twisted and laterally aligned aggregates with defined chiroptical properties. In the absence of any detectable birefringence and linear dichroism, the observed strong Cotton effect is attributed to twisted chiral amyloid superstructures. Early fibrils formed in agitated samples, but transferred to quiescent conditions before the collapse event, do not form the superstructures. On the other hand, mature insulin fibrils grown in quiescent samples and later subjected to rapid vortexing transform into clumps of finely broken fibers lacking the superstructural chirality and chiroptical properties of the continuously agitated samples. A generalized mechanism of inducing structural variants of amyloid fibrils by hydrodynamic forces favoring secondary nucleation events over elongation of fibrils is put forward. We propose that competition between low-aspect-ratio and high-aspect-ratio amyloidogenic pathways driven by fluid dynamics may play an important role in promoting distinct amyloid strains.